The present invention relates to a biologically based test system for the detection of inorganic and organic pollutants in water samples. In particular, the present invention is concerned with the development of a strategy for the control of background expression of biosensors in said biologically based test system. More particularly, the present invention comprises a biologically based test system for the detection of arsenite and arsenate in potable water with a controlled background expression of the biosensor. Furthermore, the present invention provides a test kit for determining arsenite and arsenate concentrations in water samples, said test kits are suitable for field tests.
Arsenite and arsenate are found in toxic concentrations in groundwaters in many countries all over the world and seriously threaten the life of millions of people, especially in Southeast Asian countries, like Bangladesh. In the early 1990s, high levels of arsenic in potable water of Bangladesh were first detected. Presently, about 20 million people of the total population of Bangladesh (125 million) are suspected to be drinking contaminated water. Up to a certain degree, arsenic toxicity is reversible by drinking arsenic-free water. In Bangladesh, however, arsenic-free water is not available in many places and chronic poisoning develops. The effects of arsenic poisoning can vary from skin pigmentation, development of warts, diarrhea and ulcers during the initial stages. In the most severe cases, arsenic poisoning causes liver and renal deficiencies or cancer that can lead to death (6). It is assumed that the structural similarity with phosphate causes the toxicity of arsenate. Since arsenate mimics phosphate, it can be taken up through phosphate channels and can interfere in different metabolic processes (5), (9). Arsenic poisoning is undetectable in its early stages, and takes between 8 and 14 years to have a clear impact on human health. The final impact is depending on the amount of arsenic ingested, the nutritional status and the immune response of the individual.
More than ten years ago, most of Bangladesh's population obtained its potable water from surface ponds, but this was bacteriologically unsafe and, therefore, an undesired situation. Because of these reasons, the use of groundwater as source for potable water was promoted. It is estimated that 95% or more of Bangladeshis now use groundwater for drinking water. Unfortunately, the use of groundwater for drinking water has caused unsuspected dreadful effects, due to very high concentrations of arsenic. The content of arsenic in Bangladesh' groundwater is naturally determined.
The drinking water limit for As-contamination in Bangladesh is 50 μg/l. The World Health Organization, however, recommends an index of 10 μg/l. The highest concentrations of arsenic found in Bangladesh are around 2.4 mg/l. Arsenic in potable water is not only a problem in Bangladesh, but also in other countries like India, Mexico, Vietnam or the Republic of Yugoslavia. Measuring the concentrations of arsenite and arsenate in water used as drinking water is therefore an absolute necessity. Accurate determination of arsenic compounds is usually done by Atomic Fluorescence Spectroscopy or Atomic Adsorption Spectroscopy, but both techniques require substantial investments and are not available in rural and poorer areas. Chemical field tests exist as well, but these are not accurate in the range of the present drinking water standards (10 to 50 microgram per liter) and themselves give rise to pollution with heavy metals like mercury and zinc. Moreover, the measurement of arsenite by traditional analytical chemical methods is difficult and costly (23). Thus, there is a strong interest to produce chemical field test kits, which are sensitive enough to detect arsenite around the drinking water limit of 50 μg/l. Most available tests use hydrochloric acid and zinc to produce arsine-gas (AsH3), which is then reacting with mercuric bromide on a paper, producing a brown color. However, this test is still unreliable at concentrations below 150 μg/l.
Alternative measuring strategies could involve an assay with whole cell living biosensors. Such assays have already been described in the art. In this type of biosensor assay microorganisms are used as specific and sensitive devices for sensing the bioavailability of a particular pollutant or pollutant class (21). The idea of sensing is based on the ability of pollutants to invoke a specific and inducible response in microorganisms. The signaling pathway thus activated will regulate the expression of one or more genes. The extent of this gene expression serves as a measure of the available (“sensed”) concentration of the compound. A rapid and sensitive way to measure such gene expression is to fuse relevant promoter sequences to reporter genes, like those coding for bacterial luciferase, beta-galactosidase or green flurorescent protein (GFP).
A bacterial bioassay has been described (16, 17, 19, 22 and 23), which is based on the natural resistance mechanisms of bacteria against arsenic. This assay makes use of biosensors, which are able to detect arsenite and arsenate in water samples. Said biosensors are usually genetically modified bacteria, comprising a gene coding for a regulatory protein inducing the production of a specific marker protein in the presence of arsenic. The biosensors are remarkable sensitive and detect arsenite at concentrations of 5 microgram per liter. The activity of the marker protein is usually measured by a luminometer.
However, bioassays developed so far are afflicted with high background expressions of the marker proteins, which lead to false positive results at arsenite concentrations in the microgram per liter range and this has limited until now the development of a simple field colorimetric test. Background expression occurs in all arsenic-based biosensors due to the nature of the DNA-binding of the regulatory protein.
Therefore, an urgent need exists for the development and availability of a simple, cheap, reliable, and accurate test system with a controlled background expression of the biosensor, which is suitable as a field test for the measurement of heavy metals, such as arsenic in water samples.